GB2335490A - Assay surface comprising a biotin complex - Google Patents

Assay surface comprising a biotin complex Download PDF

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Publication number
GB2335490A
GB2335490A GB9806055A GB9806055A GB2335490A GB 2335490 A GB2335490 A GB 2335490A GB 9806055 A GB9806055 A GB 9806055A GB 9806055 A GB9806055 A GB 9806055A GB 2335490 A GB2335490 A GB 2335490A
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Prior art keywords
biotin
analyte
assay
avidin
folate
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GB9806055A
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GB2335490A8 (en
GB9806055D0 (en
GB2335490B (en
Inventor
Steve Edwards
Joanna Sefton
Jayne Beverley Hipkiss
Heather Anne Edgar
Adrian Charles Dawkes
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Ortho Clinical Diagnostics Ltd
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Ortho Clinical Diagnostics Ltd
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Priority to GB9806055A priority Critical patent/GB2335490B/en
Publication of GB9806055D0 publication Critical patent/GB9806055D0/en
Priority to CA002264448A priority patent/CA2264448C/en
Priority to US09/268,796 priority patent/US20020037536A1/en
Priority to JP07325299A priority patent/JP4455688B2/en
Priority to DE69912016T priority patent/DE69912016T2/en
Priority to AT99302129T priority patent/ATE252237T1/en
Priority to EP99302129A priority patent/EP0943919B1/en
Priority to DK99302129T priority patent/DK0943919T3/en
Publication of GB2335490A publication Critical patent/GB2335490A/en
Publication of GB2335490A8 publication Critical patent/GB2335490A8/en
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Publication of GB2335490B publication Critical patent/GB2335490B/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

An assay surface comprises a carrier, coated with a molecule (preferably a polyamine, especially polylysine) coupled to biotin (or a mimic thereof), thereby to form a complex, wherein said complex is resistant to analyte release. The biotin may be bonded to avidin or streptavidin. The surface may be used in an assay of an analyte, preferably vitamin B12 or folate, which involves an analyte release, wherein said assay and said release may be performed on the same surface.

Description

1 2335490 An Assay Surface That Permits An Analyle Releasing Step
Background of the Invention
The present invention relates to a surface or solid phase for use in an assay. The present invention also relates to a method of performing an assay using said surface.
The detection and quantification of various analytes in a sample of body fluid using immunoassay and other binding protein assay techniques has been well established in fields involving the diagnosis, treatment and monitoring of disease. Analytes can be any of various factors such as proteins, hormones, chemicals and their meabolic products. A commonly used binding protein assay technique involves the immobilization of a receptor for the analyte on a reaction vessel, for example a microwell. The immobilization can be accomplished by physical adsorption, chemical coupling or by utilizing specific binding pairs such as biotin and avidin [US Patent No. 5,367,624]. Some samples require pretreatment in order to release the analyte in a form that will allow the interaction of the analyte with the analyte-specific active surface and thus enable detection of the analyte. Routinely, assays where harsh sample pretreatment for releasing an analyte is required are performed in two separate vessels or on two physically separate surfaces. Hereinafter, such a pretreatment step is referred to as an analyte releasing step.
For example, in order to measure vitamin B1 2 or folate in a serum or plasma sample, it is necessary to release any proportion of the analyte that might be complexed to a binding or carrier protein or other agent such that the analyte specific assay components can bind the analyte in question and appropriate measurements can be made. Release of the bound or complexed analyte is routinely achieved by performing an analyte releasing step, for example, by adding a strong alkali to the sample or boiling the sample in the 2 presence of a reducing agent, carried out in advance of the assay. [Branagan 1 (1991)].
A problem that arises, however, is that the reagent used in the analyte releasing step may react with an active component on the microwell surface resulting in a decrease or elimination of analyte binding. Therefore, sample pretreatments or analyte releasing steps are routinely carried out in a separate vessel using an uncoated or non-activated vessel and a proportion of sample transferred to the active reaction vessel after the analyte releasing step, thus requiring an extra step in the assay procedure. Alternatively, the assay may be performed on a separate surface which is introduced into the sample holding vessel after releasing of the analyte.
Summary of the Invention
One object of the present invention is to provide an activated surface that is resistant to an analyte releasing step. The present invention therefore eliminates the need for separate vessels or surfaces and permits carrying out an analyte releasing step, which would otherwise denature or degrade the active components on the assay surface. Therefore, the present invention relates to an assay surface, comprising: a support coated with a molecule coupled to biotin or a biotin mimic to form a complex, wherein the complex is resistant to an analyte releasing step. Preferably the molecule that is coupled to biotin or biotin mimic is a polyamine. Most preferably the polyamine is polylysine.
In another embodiment of the invention, the biotin or biotin mimic is further coupled to a biotin binding molecule selected from the group consisting of avidin and streptavidin. Either avidin or streptavidin can be used and one skilled in the art would know which one to employ depending on the assay conditions. An example of this embodiment would be a solid phase coated with polylysine coupled to biotin coupled to avidin.
Applicants' invention also relates to a method for determining the presence of an analyte in a sample wherein an analyte releasing step is 3 employed, comprising providing a surface comprising a support coated with a molecule coupled to biotin or a biotin mimic to form a complex, wherein the complex is resistant to the analyte releasing step.
Detailed Description of The Invention
According to the present invention, an assay surface is coated with a molecule, for example, a protein or polysaccharide, which is chemically coupled to biotin or a biotin mimic by standard coupling chemistry. The resulting complex can be further coupled to a biotin binding molecule such as avidin or streptavidin. For example, the avidin or streptavidin could be overcoated onto the solid phase or could be present in solution in an assay reagent. An assay could then be carried out where one or more analyte-specific components of the assay has been biotinylated.
The invention further relates to a method for an assay using such a surface. As stated above, a commonly used binding protein assay technique involves the immobilization of a receptor for an analyte on a solid support. A typical technique might then comprise contacting the solid phase with a sample that may contain the analyte, contacting the solid phase with a second binding ligand for the analyte wherein said second ligand is labeled directly or indirectly with a detectable group and measuring the amount of the detectable group bound to the solid phase. Alternatively, the amount of detectable group not bound to the solid phase can be measured as an indication of the presence of the analyte. The detectable group can be, for example, an enzyme, a radioactive atom, a fluorescent molecule or a luminescent molecule. It will be understood by one of ordinary skill in the art that above-steps can be done sequentially or simultaneously.
The assay surface can be any support, for example, a microwell, gel, membrane, particles, beads or a dipstick. One skilled in the art would know what type of support best suits the assay to be performed. Coating techniques are known in the art and include, for example, physical adsorption or chemical 4 binding. The term polyamine means a polymeric compound containing pendant amine groups, for example polylysine. A biotin mimic is any compound capable of binding to avidin or streptavidin. The high affinity interaction of biotin and avidin or streptavidin and its use in immunoassay and other immunologic techniques are well known in the art [Bayer, E.A. and Wilchek, M. (1988)].
The effectiveness and advantages of the invention are further illustrated by the following examples.
Example 1 Coating of microwells with Polyl sine - biotinlavidin In one embodiment of the invention, the polyamine used to coat the microwells is polylysine. A polylysine-biotin conjugate, prepared by reaction of poly-L-lysine hydrochloride with biotin-XX-NHS (Calbiochem, Beeston, U.K.), was added to a polystyrene microwell at 2.5mgImi- in 0.1M sodium phosphate buffer pH 7.0 or 0.1 M carbonatelbi carbonate buffer pH 10.0. The microwell was incubated at room temperature for sufficient time for the polylysine- biotin to saturate the surface. in this experiment, 6 minutes or greater was found to be sufficient. After washing the microwell, avidin was added at a concentration of 10mgImi- ia the same phosphate or carbonate buffer. The microwell was incubated with the avidin solution for sufficient time to saturate the available biotin on the microwell. In this case, 50 minutes or greater was found to be sufficient. Concentrations of polyamine-biotin and avidin are used such that the resultant biotin binding capacity of the surface is 0.05 - 0.2 ngImM2. Biotin binding capacities can be measured by methods known in the art (US Patent 5,362, 624). Avidin coated surfaces or reaction vessels prepared in this way can be used for assays where one or more of the specific assay components is labeled with biotin.
Example 2 Folate Assay using Polylysine-Biotin (PLB)/Avidin and Avidin Microwells AMERLITE polystyrene microwells were coated with polylysine-biotin (PLB) and avidin as described in Example 1. Microwells, coated only with avidin, with a similar biotin binding capacity to the P1-13-avidin coated wells, were compared as a control.
Sample Treatment: 10pi of serum samples, containing different concentrations of folate, and 1 Opt of sample treatment solution, containing 1 % ascorbic acid and 0.03% dithiothreitol (M) were added to wells and incubated for 15 minutes at 370C with shaking on an AMERLITE Incubator. Following this, 10pi of 0.8M NaOH (denaturant solution) was added and the wells incubated for a further 5 minutes at 370C. Note that all treatment and denaturant solutions are aqueous solutions.
Folate Assay: After this incubation, 100pi of 0.04mglmL biotinylated folate binding protein (biotin-FBP) and 50pi of 3ngImL folate labeled with horseradish peroxidase (folate-HRP) in borate buffers (pH 7.2) were added and the microwells were incubated for 60 minutes at 370C. Folate in a given sample will compete with folate-HRIP for binding by biotin-F13P. The biotinF13P/folate and biotin-FBP/folate-HRP complexes are immobilized on the microwell via the biotin avidin link. In the absence of foiate in a sample, all the folate bound by biotin-F13P will be labeled with HRP, so that maximum HRIP activity is immobilized on the microwell. As the concentration of folate in a given sample increases there is increasing competition with the folate-HRIP for binding by biotin-FBP, such that the higher the folate concentration in the sample, the lower the HRIP activity immobilized on the microwell.
After washing on an AMERLITE Washer, AMERLITE Signal Reagent was added to each well and the light output determined by an AMERLITE Analyzer. The HRIP activity is measured by an enhanced luminescence reaction [Whitehead et at. (1983fl. AMERLITE Signal Reagent, containing luminogenic substrates (a luminol derivitive and a peracid salt) and an enhancer, is added to the wells to initiate the light emitting reaction. The purpose of the enhancer (a 6 substituted phenol) is to increase the level of light produced and prolong its emission. The results of the assay are given below in Table 1.
Table 1:
Folate Mean Signal Level (arbitrary light units) Concentration (ngImL) PLB/Avidin Wells Control Wells 0.0 22.0 11.5 0.25 19.9 11.6 0.5 21.3 11.9 0.75 19.3 11.9 1.0 19.0 11.5 1.5 17.4 11.8 2.0 17.7 11.9 3.0 15.8 10.6 4.0 14.1 9.3 5.0 12.2 8.9 Example 3 Vitamin B12 Assay using Polylysine-Biotin (PLB)/Avidin and Avidin MicroWells AMERLITE polystyrene microwells were coated with polylysine-biotin (PLB) and avidin as described in Example 1. Microwells, coated only with avidin, with a similar biotin binding capacity to the P1-13-avidin coated wells, were compared as a control.
Sample Treatment: 10pi of serum samples, containing different concentrations of vitamin B12, and 20pi of sample treatm ent/denatu rant solution containing 0.015 % KCN, 0.1% dithiothreitol (DTT) and 0.5M NaOH were added to wells and incubated for 15 minutes at 370C with shaking on an AMERLITE Incubator.
7 Vitamin B12 Assay: After this incubation, 100pi of 5ngImL biotinylated intrinsic factor (biotin-IF) and 50p] of 2ngImL vitamin B12 labeled with horseradish peroxidase (vitamin B12-HRP) in phosphate buffer (pH 6. 4) were added and the microwells incubated for 60 minutes at 370C. Vitamin B12 in a given sample will compete with vitamin B12-HRP for binding by biotin- IF.
The biotin-1171vitamin B12 and biotin-1171vitamin B12-HRP complexes are immobilized on the solid phase via the biotin avidin link. In the absence of vitamin B12 in a sample, all the vitamin B12 bound by biotin-IF will be labeled with HRIP so that maximum HRIP activity is immobilized on the microwell. As the concentration of vitamin B12 in a given sample increases there is increasing competition with the vitamin B12-HRP for binding by biotin-IF such that the higher the concentration in the sample, the lower the HRP activity immobilized on the microwell.
After washing on an AMERLITE Washer, AMERLITE Signal Reagent was added to each well and the light output determined by an AMERLITE Analyzer.
The HRP activity is measured by an enhanced luminescence reaction as in Example 2. The results of the assay are given below in Table 2.
Table 2:
Vitamin B12 Conc. Mean Signal Level (arbitrary light units) (pg/M1) PLB/Avidin Wells Control Wells 0 11.61 6.94 10.29 6.47 7.21 4.15 396 4.06 2.45 898 2.67 1.52 1838 1.19 0.48 8 Results of Folate and Vitamin B1 2 Assays The results, given in Tables 1 and 2, show that the use of PL13/avidin coated microwells gives significantly greater signal in the absence of folate and Vitamin B12 and a larger decrease in signal as the concentration of analyte increases compared to the avidin control wells. The differences seen are attributable to loss of biotin-binding capacity in the control wells following the sample denaturation treatment.
Cited Literature 1. U.S. Patent No. 5,367,624.
2. Branagan, P., Kit Inspection Laboratory Practice 40:11, pp. 27-30 (1991). 3. Christenson, R. H., Dent, G.A. and Tuszyriski, A, "Two Radioassays for Serum Vitamin B12 and Folate Determination Compared in a Reference Interval Study," Clin. Chem. 31:8, pp. 1358-1360 (1985).
4. Bayer, E.A. and Wilchek, M. "The Avidin-Biotin Complex in Bioanalytical Applications," AnaL Chem. vol. 171, pp. 1-32 (1988). 5. Whitehead, T.P. et a[., "Enhanced Luminescence Procedure For Sensitive Determination of Peroxidase-labeled Conjugates In Immunoassay,', Nature vol. 305, pp. 158-9 (1983).
All cited materials herein are hereby incorporated by reference.
Accordingly, it should be noted that the present invention includes all modifications failing within the scope of the following claims. It is to be understood that numerous changes and modifications may be made therein without departing from the scope and intent of the invention.
9

Claims (8)

1. An assay surface, comprising a support coated with a molecule coupled to biotin or a biotin mimic to form a complex, wherein the complex is resistant to 5 an analyte releasing step.
2. The surface of claim 1, wherein the molecule is a polyamine.
3. The surface of claim 2, wherein the polyamine is polylysine.
4. The surface of any one of claims 1 to 3, wherein the biotin or biotin mimic is further coupled to a biotin binding molecule selected from the group consisting of avidin and streptavidin.
5. A method for an assay for determining the presence of an analyte in a sample wherein an analyte releasing step is employed, comprising: providing a surface comprising a support coated with a molecule coupled to biotin or a biotin mimic to form a complex, wherein the complex is resistant to the analyte releasing step. 20
6. The method of claim 5 wherein the analyte is vitamin B12.
7. The method of claim 5 wherein the analyte is folate.
8. The method of any one of claims 5 to 7 wherein the analyte releasing step and the assay are performed on the same surface.
GB9806055A 1998-03-20 1998-03-20 An assay surface that permits an analyte releasiing step Revoked GB2335490B (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
GB9806055A GB2335490B (en) 1998-03-20 1998-03-20 An assay surface that permits an analyte releasiing step
CA002264448A CA2264448C (en) 1998-03-20 1999-03-05 An assay surface that permits an analyte releasing step
US09/268,796 US20020037536A1 (en) 1998-03-20 1999-03-16 An assay surface that permits an analyte releasing step
JP07325299A JP4455688B2 (en) 1998-03-20 1999-03-18 An assay surface that allows an analyte release step
EP99302129A EP0943919B1 (en) 1998-03-20 1999-03-19 An assay surface that permits an analyte releasing step
AT99302129T ATE252237T1 (en) 1998-03-20 1999-03-19 A TEST SURFACE THAT ALLOWS AN ANALYTE BINDING STEP
DE69912016T DE69912016T2 (en) 1998-03-20 1999-03-19 A test surface that allows an analyte release step
DK99302129T DK0943919T3 (en) 1998-03-20 1999-03-19 Test surface enabling an analyte releasing step

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Application Number Priority Date Filing Date Title
GB9806055A GB2335490B (en) 1998-03-20 1998-03-20 An assay surface that permits an analyte releasiing step

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GB9806055D0 GB9806055D0 (en) 1998-05-20
GB2335490A true GB2335490A (en) 1999-09-22
GB2335490A8 GB2335490A8 (en) 2000-03-08
GB2335490B GB2335490B (en) 2003-05-14

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GB9806055A Revoked GB2335490B (en) 1998-03-20 1998-03-20 An assay surface that permits an analyte releasiing step

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EP (1) EP0943919B1 (en)
JP (1) JP4455688B2 (en)
AT (1) ATE252237T1 (en)
CA (1) CA2264448C (en)
DE (1) DE69912016T2 (en)
DK (1) DK0943919T3 (en)
GB (1) GB2335490B (en)

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Publication number Priority date Publication date Assignee Title
FR2814463B1 (en) * 2000-09-22 2002-11-15 Sanofi Synthelabo NOVEL POLYSACCHARIDES WITH ANTITHROMBOTIC ACTIVITY COMPRISING AT LEAST ONE COVALENT BINDING WITH BIOTIN OR A BIOTINE DERIVATIVE
ES2564293T5 (en) * 2002-02-28 2019-04-04 Microsens Biophage Ltd Enlazamiento of pathological forms of prion proteins
CN105861676B (en) * 2016-04-27 2020-03-17 郑州科蒂亚生物技术有限公司 Buffer solution for coating nucleic acid primer and preparation method of coated nucleic acid primer
CN113759107A (en) * 2021-08-18 2021-12-07 苏州立禾生物医学工程有限公司 Biotin labeling buffer solution

Citations (6)

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JPS61149863A (en) * 1984-12-24 1986-07-08 Ss Pharmaceut Co Ltd Measuring method of biotin and reagent for measurement to be used therein
WO1990004786A1 (en) * 1988-10-17 1990-05-03 Molecular Devices Corporation Hapten derivatized capture membrane and diagnostic assays using such membrane
US4935339A (en) * 1985-05-07 1990-06-19 Nichols Institute Diagnostics Delayed solid phase immunologic assay
WO1993014405A1 (en) * 1992-01-06 1993-07-22 Baxter Diagnostics Inc. Binding protein capture assay
US5252743A (en) * 1989-11-13 1993-10-12 Affymax Technologies N.V. Spatially-addressable immobilization of anti-ligands on surfaces
EP0657737A2 (en) * 1993-12-13 1995-06-14 Hewlett-Packard Company Method and reagents for binding chemical analytes to a substrate surface, and related analytical devices and diagnostic techniques

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EP0348174A3 (en) * 1988-06-23 1991-05-22 Bio-Rad Laboratories, Inc. Sperm antibody test
DK100592D0 (en) * 1992-08-10 1992-08-10 Mouritsen & Elsner Aps METHOD OF CHEMICAL CONNECTION ON SOLID PHASES
US5629213A (en) * 1995-03-03 1997-05-13 Kornguth; Steven E. Analytical biosensor
DE19530078A1 (en) * 1995-08-16 1997-02-20 Bayer Ag Optical solid phase biosensor based on streptavidin and biotin
GB9719140D0 (en) * 1997-09-09 1997-11-12 Ortho Clinical Diagnostics Ligand binding sufaces

Patent Citations (6)

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Publication number Priority date Publication date Assignee Title
JPS61149863A (en) * 1984-12-24 1986-07-08 Ss Pharmaceut Co Ltd Measuring method of biotin and reagent for measurement to be used therein
US4935339A (en) * 1985-05-07 1990-06-19 Nichols Institute Diagnostics Delayed solid phase immunologic assay
WO1990004786A1 (en) * 1988-10-17 1990-05-03 Molecular Devices Corporation Hapten derivatized capture membrane and diagnostic assays using such membrane
US5252743A (en) * 1989-11-13 1993-10-12 Affymax Technologies N.V. Spatially-addressable immobilization of anti-ligands on surfaces
WO1993014405A1 (en) * 1992-01-06 1993-07-22 Baxter Diagnostics Inc. Binding protein capture assay
EP0657737A2 (en) * 1993-12-13 1995-06-14 Hewlett-Packard Company Method and reagents for binding chemical analytes to a substrate surface, and related analytical devices and diagnostic techniques

Non-Patent Citations (1)

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Title
Derwent WPI Abstract Accession No. 86-216851/198633 & JP 61-149863 A *

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CA2264448A1 (en) 1999-09-20
EP0943919A1 (en) 1999-09-22
GB2335490A8 (en) 2000-03-08
GB9806055D0 (en) 1998-05-20
EP0943919B1 (en) 2003-10-15
ATE252237T1 (en) 2003-11-15
CA2264448C (en) 2008-10-28
DE69912016T2 (en) 2004-08-19
US20020037536A1 (en) 2002-03-28
JP4455688B2 (en) 2010-04-21
JPH11311624A (en) 1999-11-09
GB2335490B (en) 2003-05-14
DK0943919T3 (en) 2004-02-16
DE69912016D1 (en) 2003-11-20

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